• Molecules and Cells
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• 제39권10호
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• pp.750-755
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• 2016

Although innate color preference of motile organisms may provide clues to behavioral biases, it has remained a longstanding question. In this study, we investigated innate color preference of zebrafish larvae. A cross maze with different color sleeves around each arm was used for the color preference test (R; red, G; green, B; blue, Y; yellow). The findings showed that 5 dpf zebrafish larvae preferred blue over other colors (B > R > G > Y). To study innate color recognition further, tyrosinase mutants were generated using CRISPR/Cas9 system. As a model for oculocutaneous albinism (OCA) and color vision impairment, tyrosinase mutants demonstrated diminished color sensation, indicated mainly by hypopigmentation of the retinal pigment epithelium (RPE). Due to its relative simplicity and ease, color preference screening using zebrafish larvae is suitable for high-throughput screening applications. This system may potentially be applied to the analysis of drug effects on larval behavior or the detection of sensory deficits in neurological disorder models, such as autism-related disorders, using mutant larvae generated by the CRISPR/Cas9 technique.

• Molecular & Cellular Toxicology
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• 제1권3호
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• pp.179-188
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• 2005

To develop the novel anti-allergic drug, many sophoricoside derivatives were synthesized. Among these derivatives, JSH-II-3, VI-3, VII-3, VIII-3, VII-20 and VII-20 (sodium salt) were selected and subjected to high throughput toxicity screening (HTTS) because they revealed strong IL-5 inhibitory activity and limitation of quantity. Single cell gel electrophoresis (Comet) assay, mouse lymphoma thymidine kinase ($tk^{+/-}$) gene assay (MOLY), chromosomal aberration assay in mammalian cells and Ames reverse mutation assay in bacterial system were used as simplified, inexpensive, short-term in vitro screening tests in our laboratory. Through the primary screening using the comet assay, we could choose the first candidates of sophoricoside derivatives with no genotoxic potentials as JSH-VI-3, VII-3, VII-20 and VII-20 (sodium salt). Also JSH-VII-3, VII-20 and VII-20 (sodium salt) are non-mutagenic in MOLY assay, while JSH-II-3 is mutagenic at high concentration with the presence of metabolic activation system in both comet assay and MOLY assay. The selected derivatives (JSH-VI-3, VII-3, VII-20 and VII-20 (sodium salt) are not mutagenic in S. typhimurium TA98 and TA100 strains both in the presence and absence of metabolic activation. From results of chromosomal aberration assay, 6 h treatment of JSH-VI-3, VII-3 and VII-20 (sodium salt) were not revealed clastogenicity both in the presence and absence of S-9 mixture. Therefore, we suggests that JSH-VI-3, VII-3, VII-20 and VII-20 (sodium salt), as the optimal candidates with both no genotoxic potential and IL-5 inhibitory effects must be chosen. To process the development into new anti-inflammatory drug of these derivatives, further investigation will need.

• Journal of Genetic Medicine
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• 제12권1호
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• pp.25-30
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• 2015

Purpose: Charcot-Marie-Tooth disease (CMT) is a peripheral neuropathy mainly divided into CMT type 1 (CMT1) and CMT2 according to the phenotype and genotype. Although molecular pathologies for each genetic causative have not been revealed in CMT2, the correlation between cell death and accumulation of misfolded proteins in the endoplasmic reticulum (ER) of Schwann cells is well documented in CMT1. Establishment of in vitro models of ER stress-mediated Schwann cell death might be useful in developing drug-screening systems for the treatment of CMT1. Materials and Methods: To develop high-throughput screening (HTS) systems for CMT1, we generated cell models using transient expression of mutant proteins and chemical induction. Results: Overexpression of wild type and mutant peripheral myelin protein 22 (PMP22) induced ER stress. Similar results were obtained from mutant myelin protein zero (MPZ) proteins. Protein localization revealed that expressed mutant PMP22 and MPZ proteins accumulated in the ER of Schwann cells. Overexpression of wild type and L16P mutant PMP22 also reduced cell viability, implying protein accumulation-mediated ER stress causes cell death. To develop more stable screening systems, we mimicked the ER stress-mediated cell death in Schwann cells using ER stress inducing chemicals. Thapsigargin treatment caused cell death via ER stress in a dose dependent manner, which was measured by expression of ER stress markers. Conclusion: We have developed genetically and chemically induced ER stress models using Schwann cells. Application of these models to HTS systems might facilitate the elucidation of molecular pathology and development of therapeutic options for CMT1.

• Biomolecules & Therapeutics
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• 제22권5호
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• pp.371-383
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• 2014

The nematode Caenorhabditis elegans (C. elegans) offers a unique opportunity for biological and basic medical researches due to its genetic tractability and well-defined developmental lineage. It also provides an exceptional model for genetic, molecular, and cellular analysis of human disease-related genes. Recently, C. elegans has been used as an ideal model for the identification and functional analysis of drugs (or small-molecules) in vivo. In this review, we describe conserved oncogenic signaling pathways (Wnt, Notch, and Ras) and their potential roles in the development of cancer stem cells. During C. elegans germline development, these signaling pathways regulate multiple cellular processes such as germline stem cell niche specification, germline stem cell maintenance, and germ cell fate specification. Therefore, the aberrant regulations of these signaling pathways can cause either loss of germline stem cells or overproliferation of a specific cell type, resulting in sterility. This sterility phenotype allows us to identify drugs that can modulate the oncogenic signaling pathways directly or indirectly through a high-throughput screening. Current in vivo or in vitro screening methods are largely focused on the specific core signaling components. However, this phenotype-based screening will identify drugs that possibly target upstream or downstream of core signaling pathways as well as exclude toxic effects. Although phenotype-based drug screening is ideal, the identification of drug targets is a major challenge. We here introduce a new technique, called Drug Affinity Responsive Target Stability (DARTS). This innovative method is able to identify the target of the identified drug. Importantly, signaling pathways and their regulators in C. elegans are highly conserved in most vertebrates, including humans. Therefore, C. elegans will provide a great opportunity to identify therapeutic drugs and their targets, as well as to understand mechanisms underlying the formation of cancer.

• 한국육종학회지
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• 제43권4호
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• pp.233-240
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• 2011

식물 피노믹스 분야에서 초고속 대량선발이 가능하도록 만든 화상기술(imaging technology)을 온실자동화 기술, 이미지 촬영 및 분석기술 등으로 분류하여 개념을 정리하고, 화상기술을 개발 및 응용하고 있는 주요 연구기관의 현황을 소개하였다. 연구동향 파악을 위해 작물의 내재해성 검정, 병해충진단, 종자활력 검정, 수확후 관리, 생체리듬 연구 등 다양한 분야에서 응용되고 있는 사례들을 살펴보았다. 향후 열 화상, 형광 화상 기술을 UV-induced blue green fluorescence, hyperspectral imaging 등과 상호 보완해서 multi-sensor 개념으로 발전시켜 나간다면, 식물의 생산량 증대를 위한 스트레스 내성자원 선발의 효율성을 극대화할 수 있을 뿐만 아니라 다양한 파장대의 영상정보로부터 각 스트레스의 특징을 catalogue화하는 것이 가능하여 다양한 스트레스를 정확히 진단하고 정량화할 수 있을 것으로 보이며, 나아가 각종 스트레스에 대한 조기 경보시스템으로도 활용할 수 있을 것이다. 호주 Plant Phenomics Centre에서는 온실과 포장을 포함한 Phenomics 기술의 종합적 개념도를 그림 4와 같이 제시하고 각 부문별 필요기술을 개발 중에 있는데, 종합기술로 완성된다면 현재의 작물 품종개량 속도를 획기적으로 향상시킬 수 있을 것으로 기대된다. 호주는 그 동안 분자생물학, 식물생리학 분야에서는 기술력을 확보해 왔지만 BT, IT 등 융복합농업기술분야에서는 다른 나라에 비해 역량을 결집하지 못하고 뒤쳐져 있었다고 볼 수 있는데, 연구개발의 궁극적인 목표인 실용화 단계에서의 기술우위를 선점함으로써 그간의 약세를 만회하고자 Phenomics 연구시설을 설치하였다고 한다. 이점은 BT 후발주자이면서 국제적 경쟁력을 확보하려는 우리에게 시사하는 바가 크다. Phenomics 연구는 생명공학기술을 통해 창출된 GM 식물체, 전통육종을 통해 육성된 육종재료 등 모든 유용 유전자원을 평가, 검정할 수 있는 신품종 육성의 기본 기술로 부상하고 있다. 지난 수년간 정체되어 있는 국내 종자시장을 고부가 수출산업으로 육성하기 위해서는 유용 유전자원, 농업생명공학산물의 실용화를 가속화하여야 하며, 이를 위해 피노믹스 기술 확보 및 시설 인프라 구축을 전략적으로 신중히 검토해봐야 할 시점이다.

• BMB Reports
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• 제38권5호
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• pp.517-525
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• 2005

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.

• 대한화장품학회지
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• 제31권1호
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• pp.13-16
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• 2005

미백 물질 탐색 방법으로, MC1R 발현 인자인 Mitf (microrhthalmia transcription factor)를 이용한 protein chip을 적용하였다. MC1R promoter와 Mitf 결합의 저해 인자로써, DNA 상의 결합 부위인 E-box (CATGTG)와 유사한 서열을 가진 oligomer를 사용하였고, E-box 내외부의 서열 변화에 따른 저해율 또한 측정하였다. 그 결과 DNA-Mitf 결합 저해율에 있어서, E-box 서열 내 변화를 준 oligonucleotide 경쟁자는, E-box 이외의 서열 변화를 준 경쟁자보다 낮은 수치를 보였다.

• 한국미생물·생명공학회지
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• 제47권1호
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• pp.69-77
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• 2019

Screening microorganisms that can produce glucan hydrolases for industrial, environmental, and biomedical applications is important. Herein, we describe a novel approach to perform glucan hydrolase screening-based on analysis of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) spectra-which involves degradation of the oligo- and polysaccharides. As a proof-of-concept study, glucan hydrolases that could break down glucans made of several glucose units were used to demonstrate the MALDI-MS-based enzyme assay. First, the enzyme activities of ${\alpha}$-amylase and cellulase on a mixture of glucan oligosaccharides were successfully discriminated, where changes of the MALDI-MS profiles directly reflected the glucan hydrolase activities. Next, we validated that this MALDI-MS-based enzyme assay could be applied to glucan polysaccharides (i.e., pullulan, lichenan, and schizophyllan). Finally, the bacterial glucan hydrolase activities were screened on 96-well plate-based platforms, using cell lysates or samples of secreted enzyme. Our results demonstrated that the MALDI-MS-based enzyme assay system would be useful for investigating bacterial glucoside hydrolases in a high-throughput manner.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2005년도 국제학술심포지움 The 44th Annual Meeting of Korean Society for Life Science
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• pp.169-170
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• 2005

• 한국전기전자재료학회논문지
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• 제17권4호
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• pp.404-409
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• 2004

We fabricated photo-sensor for fluorescence detection in LOC. LOC is high throughput screening system. Our LOC screens biochemical reaction of protein using the immunoassay, and converts biochemical reaction into electrical signal using LIF(Laser Induced Fluorescence) detection method. Protein is labeled with rhodamine intercalating dye and finger PIN photodiode is used as photo-sensor We measured fluorescence emission of rhodamine dye and analyzed tendency of fluorescence detection, according to photo-sensor size, light intensity, and rhodamine concentration. Detection current was almost linearly proportional to two parameters, intensity and concentration, and was inversely proportional to photo-sensor size. Integrated LOC consists of optical-filter deposited photo-sensor and PDMS microchannel detected 50 (pg/${mu}ell$) rhodamine. For integrated LOC including light source, we used green LED as the light source and measured emitted fluorescence.